Estudo da interac����o de complexos de van��dio com a ca2+-atpase de ret��culo sarcoplasm��tico: an��lise estrutural e funcional

A bomba de c��lcio de ret��culo sarcoplasm��tico �� uma das prote��nas mais extensivamente estudadas, capaz de interagir com v��rias esp��cies e compostos de van��dio. Combinando-se estudos de fluoresc��ncia com ensaios cin��ticos de transporte e liga����o de 45Ca �� ATPase, avaliou-se o efeito de tr��s complexos de van��dio na fun����o bioenerg��tica e estrutural de Ca2+-ATPase. Demonstrou-se que concentra����es pr��ximas dos valores de IC50 (para a hidr��lise de ATP) de BMOV-V(IV) e de PDC-V(V) n��o inibem significativamente a acumula����o de 45Ca por sarcoves��culas. Por outro lado, o complexo PDC-V(V) mostrou ser capaz de estimular a liga����o de 45Ca ao ret��culo sarcoplasm��tico, sugerindo que este complexos pode interagir com o dom��nio de liga����o de c��lcio �� bomba. V��rios estudos de fluoresc��ncia mostraram que BMOV-V(IV) e PDC-V(V) poder��o ser capazes de induzir a conforma����o E2 (tal como solu����es de ���decavanadato��� e de ���monovanadato���) e E1 de Ca2+-ATPase (tal como solu����es de ���metavanadato���), respectivamente. Apesar de o composto HAIDA-V(IV) previlegiar a conforma����o E1 (devido �� sua elevada afinidade para i��es Ca2+), inibiu significativamente o transporte e a liga����o de 45Ca, o que sugere que possa interagir com os locais de uni��o de c��lcio. Os resultados obtidos s��o consistentes com a forma����o de um aducto entre o composto de van��dio e a prote��na, o que sugere um efeito na homeostasia intracelular de c��lcio, nos sistemas de contrac����o muscular e, inclusive, nas vias de ac����o de insulina. Cada um dos tr��s complexos promoveu respostas distintas na Ca2+-ATPase, sugerindo uma evidencia de actividades biol��gicas diversas em fun����o da esp��cie qu��mica de V e do ambiente de coordena����o.

Ligand binding and signalling pathways of PTH receptors in sea bream (Sparus auratus) enterocytes

Whole animal studies have indicated that Ca2+ uptake by the gastrointestinal tract is regulated by the action of parathyroid hormone-related peptide (PTHrP) in teleost fish. We have characterised PTH receptors (PTHR) in piscine enterocytes and established, by using aminoterminal PTHrP peptides, the amino acid residues important for receptor activation and for stabilising the ligand/receptor complex. Ligand binding of 125I-(1���35tyr) PTHrP to the membrane fraction of isolated sea bream enterocytes revealed the existence of a single saturable high-affinity receptor (KD=2.59 nM; Bmax=71 fmol/mg protein). Reverse transcription/polymerase chain reaction with specific primers for sea bream PTH1R and PTH3R confirmed the mRNA expression of only the later receptor. Fugu (1���34) PTHrP increased cAMP levels in enterocytes but had no effect on total inositol phosphate accumulation. The aminoterminal peptides (2���34)PTHrP, (3���34)PTHrP and (7���34) PTHrP bound efficiently to the receptor but were severely defective in stimulating cAMP in enterocyte cells indicating that the first six residues of piscine (1���34)PTHrP, although not important for receptor binding, are essential for activation of the adenylate cyclase/phosphokinase A (AC-PKA)-receptor-coupled intracellular signalling pathway. Therefore, PTHrP in teleosts acts on the gastrointestinal tract through PTH3R and the AC-PKA intracellular signalling pathway and might regulate Ca2+ uptake at this site. Ligand-receptor binding and activity throughout the vertebrates appears to be allocated to the same amino acid residues of the amino-terminal domain of the PTHrP molecule.

Cloning of the Bone Gla Protein gene from the teleost fish Sparus aurata (gilthead seabream). Molecular organization, developmental appearance and evolutionary implications

Tese de Doutoramento, Biologia Molecular, Faculdade de Ci��ncias do Mar e do Ambiente, Universidade do Algarve, 2001